The purpose of this project is to help elucidate how HIV persists in vivo and thus to aid the development of novel HIV cure-directed therapies. The focus of the work is a small population of CD4 T cells that contain replication-competent HIV and persist at nearly stable levels even in the setting of effective antiretroviral therapy (ART). Although these cells have been considered to represent a latent reservoir, the cellular processes and underlying gene expression patterns that maintain their numbers remain incompletely defined. Indeed, it remains possible that the ongoing cell-to-cell spread of infectious virus, the proliferation of cells infected before institution of ART, and the long-term quiescence (latency) of infected cells all present barriers to cure during effective ART. Distinguishing the contributions of these mechanisms to the barrier to HIV cure in vivo will be critical because interventions designed to antagonize each individual mechanism may fail to affect or even worsen the others. Unfortunately, distinguishing the contributions of these mechanisms in blood and tissue samples from HIV-infected study participants has been extremely challenging. A major reason for this is that the CD4 T cells that contain HIV in vivo often lack expression of virus- or host-encoded markers. This prevents identification and isolation of highly pure populations of cells for the types of comprehensive downstream analysis that might reveal transcriptomic, epigenetic, or proteomic signatures of ongoing virus replication, cellular proliferation, or cellular latency. In this project, we are developing two complementary approaches to overcoming this technical challenge: a reverse approach in which phenotypic CD4 T cell subsets are sorted by flow cytometry for downstream characterization as hosts for the virus, and a forward approach in which cells containing HIV genomic DNA are isolated for comprehensive analysis following detection in water-in-oil emulsions.